WO2000078931A2

WO2000078931A2 - Neuronal cell material, use of said material as a transplant and method for producing the same

Info

Publication number: WO2000078931A2
Application number: PCT/DE2000/001932
Authority: WO
Inventors: Horst Peschel
Original assignee: Horst Peschel
Priority date: 1999-06-19
Filing date: 2000-06-19
Publication date: 2000-12-28
Also published as: DE19928210B4; EP1185625A2; EP1185625B1; ES2259608T3; DE50012777D1; AU6424900A; PT1185625E; DK1185625T3; CY1105067T1; DE19928210A1; US20060099192A1; WO2000078931A3; ATE326526T1

Abstract

The invention relates to a neuronal cell material that is suitable for transplants. According to the invention, the cell material contains no physiologically active parts on the glial cells, in order to avoid unwanted immunological side effects. The invention also relates to a method for producing a cell culture that is capable of growth, consisting of progenitor cells. Said method comprises the following steps: removing parts of the brain of a mammal, selecting progenitor cells, reproducing the progenitor cell clones and priming the progenitor cells to determined cells.

Description

Neural cell material, use as a graft and method for its production

The invention relates to a neuronal cell material, its use as a transplant in neurology or neurosurgery and a method for its production.

For the treatment of certain diseases of the nervous system, the concept is pursued of treating these diseases by transplanting suitable cell cultures. This therapy is based on the knowledge that certain diseases can be assigned to functional disorders or the death of certain types of nerve cells. It is possible to treat Parkinson's disease by transplanting dopammerger neurons, Alzheimner's disease by transplanting cholinergic neurons, Huntington's chorea by transplanting striatal neurons and MSA by transplanting stπatal and dopammerger neurons.

The problem with this therapeutic approach at the moment is that the corresponding transplants are not available to a sufficient extent, since these are mostly obtained from fetal tissue.

Another problem lies in possible rejection reactions, since different cell types are present in the graft, which can trigger tissue rejection as an immune response. For this reason, immunosuppression using suitable medicinal products is usually necessary, but these can have undesirable side effects.

In addition, there is the problem that the cells prepared in the previously known way have to be transplanted after only a few days, which increases the possibilities of an fectiological examination, for example with regard to AIDS or hepatitis viruses, are restricted.

The object of the invention is to provide neural cell material which is suitable for transplantation or from which transplants can be obtained which does not have the disadvantages mentioned above. Furthermore, the invention is based on the object of providing a method for producing such a cell material or such grafts.

The task is solved by the features of the independent claims. Advantageous further developments can be found in the subclaims.

The invention is based on the concept of keeping and multiplying progenitor cells in culture. This increase can be controlled by the action of suitable active substances, in particular proteins, such that these progenitor cells, after transplantation or generally after contacting with a suitable substrate such as cell material or a carrier material, only or predominantly differentiate into a specific cell type (e.g. dopammerge neurons). This treatment can be used to obtain cell cultures which consist practically exclusively of the direct precursors of the desired neuronal cells and, in particular, only contain immunocompetent glial cells in proportions which no longer have a physiological effect, in particular are no longer detectable. In this way, suitable grafts that are better defined with respect to the existing cell types can be produced to a practically unlimited extent.

In this way, cell material can be obtained that consists practically exclusively of dopammergeic neurons and / or cholmergic neurons and / or GABAergic striatal and / or serotonergic neurons individually or Contained in combinations, ie the proportion of the neurons mentioned in the cell material is greater than 90%, preferably greater than 95% or does not contain any physiologically active proportions of other cells, in particular glial cells.

The progenitor cells, through the multiplication and selection of which cell cultures or transplantable cell material is obtained, can be obtained from both fetal and adult neuronal cell material (brain or spinal cord). Up to now, the adult cell material has advantageously been prepared from peppticular or hippocampal sections.

The location of fetal cell removal may depend on the target tissue, i.e. Progenitor cells are removed from the areas where the desired neurons occur later, e.g. dopammerge neurons from midbrain. With suitable partial differentiation by means of the method according to the invention, however, different progenitor cells in particular can also be obtained from tissue from one and the same sampling point, e.g. also from blood stem cells from umbilical cord tissue.

The neuronal cells from fetuses with an age of 3 to 25 weeks, preferably 5 to 11 weeks, are advantageously prepared after fertilization.

The isolation and cultivation of neuronal progenitor cells from the rodent brain has been described several times (Daadi and Weiss, J. Neurosci 1999; Magrassi et al., Development 1998; 54: 107-115; Ptak et al., Cell Transplant 1995; 4: 299 -310; Liepelt et al., Brain Res Dev Brain Res 1990; 51: 267-278). The authors were able to prepare progenitor cells from different areas in the brain. Furthermore, human progenitor cells from fetal cortical tissue (or the entire brain) were prepared (Buc-Caron, Neurobiol Dis 1995; 2: 37-47; Svandsen CN et al., Exp Neurol 1997; 148: 135-146; Sah et al., Nat Biotechnol 1997; 15: 574-580; Chalmers-Redman et al., Neuroscience 1997; 76: 1121-1128). With regard to the manner in which the progenitor cells are prepared, reference is hereby made in full to the above-mentioned publications.

The provision of transplantable neuronal cell material is carried out according to the invention by a method which comprises an expansion of the indirectly or directly obtained human progenitor cells, a partial in vitro differentiation and a selection, the neuronal cultures ultimately obtained in particular, if appropriate, without the addition of other factors or permanent genetic Manipulate manipulations with a high percentage in the desired cell type.

If necessary, the cell material can be expanded again after the partial differentiation or selection of the progenitor cells.

The partial differentiation and the selection can be repeated several times, the manner in which they are carried out can differ in each case.

The invention makes it possible overall to select and differentiate semi-immature neuronal progenitor cells to such an extent that, after the addition of nutrient media or after transplantation, only or predominantly a specific cell type is discriminated.

The expansion can in particular be carried out simultaneously using the processes of hypoxia, Pπmmg, if appropriate with reduced oxygen content in the atmosphere, transient or non-transient genetic manipulation and / or treatment with exogenous factors, in particular under reduced conditions Oxygen content in the atmosphere. The process steps will be described in more detail later.

The selection can be made by subcloning, in particular by subcloning under hypoxia.

The method can in particular be carried out in such a way that, after the expansion of the freshly obtained progenitor cells, a selective expansion of the desired cell type is carried out. The selective expansion can take place by changing the oxygen content, the application of suitable mitogens or a partial differentiation (Primmg) by means of exogenous factors, optionally in each case with a reduced oxygen content in the atmosphere. The "determined" progenitors can be selected by subcloning, optionally with a reduced oxygen content in the atmosphere. Subcloning can be followed by renewed expansion and, if necessary, renewed partial differentiation by one of the methods specified above (hypoxia, Primmg, or treatment with exogenous factors), in particular by treatment with exogenous factors or by Primmg, optionally with a reduced oxygen content of the atmosphere . The first or second partial differentiation can also be carried out or supported by transient or non-transient genetic manipulation. As an alternative or in addition to a reduction in the oxygen content, the process steps can also be carried out under conditions which simulate changes induced by this changed oxygen content (e.g. with mitochondral respiratory inhibitors such as Rotenon, MPP + or Malonat).

The expansion after the individual treatments should preferably start from a single cell.

The success of selecting these determinate Progenitor cells, ie the characterization of the neuron populations obtained is carried out after complete differentiation (in vitro or in vivo), advantageously using cytometric, biochemical, molecular biological, immunohistochemical and electrophysiological methods. These can be found in the relevant specialist literature for the various neuron populations.

Expansion under hypoxic conditions:

It has been found that a considerable improvement in the division rate can be achieved by lowering the atmospheric oxygen content and / or increasing the nitrogen content, thereby simultaneously reducing the proportions of special neuronal progenitor cells, in particular the dopammerger. Neurons, are increased, so a differentiation takes place. A reduction in the oxygen voltage from 21% (indoor air) to 10%, preferably 5%, particularly preferably 1%, may be mentioned here, if appropriate in combination with a simultaneous increase in the nitrogen content, it also being possible to use other gases, for example 1- 10, preferably about 5% CO ₂ . The lowering of the atmospheric oxygen content takes place under conditions under which the cell material increases. With this technique of cultivation, mammalian, especially human, progenitor cells can also be established from the various areas of the brain (including midbrain).

As an alternative to lowering the atmospheric oxygen content, substances with normal atmospheric oxygen content can be used which impair energy production and simulate a reduced oxygen content, in particular mitochondral respiratory inhibitors (Rotenone, MPP +, etc.)

Exogenous factors used in the expansion (see below) can be used individually or in combination in concentrations from 4000 to 0.01 ng / ml, preferably 500 to 1 ng / ml, particularly preferably 100 to 2 ng / ml of the expansion solution, without being restricted to this.

Partial differentiation through Primmg:

Primmg includes treating the (monoclonal) neuronal progenitor cells with a growth factor, cytokms, neurotransmitters and / or another active ingredient, in particular one or more of the ones mentioned below, which promotes differentiation into the desired nerve cell population. So-called conditioned media can also be used for this purpose, i.e. Culture media which are used for the cultivation of, in particular, the neurons of the target area of the desired nerve cell population (e.g. stπatum in the case of dopammergenic neurons) or glial cells or which are obtained from primary cultures of these cells. These media contain the proteins that were secreted by the respective cells. These exogenous factors are then withdrawn at a time (preferably after a few hours) at which the cells again dedifferentiate in a state in which further expansion is possible. Progenitor cells converted in this way are referred to as primed cells. With a second exposure to effective endogenous factors (e.g. after transplantation into an adult brain) the differentiation is much faster. In the case of (renewed) expansion, primed (possibly also monoclonal) cell lines are obtained which already expand genes which ensure a higher specificity. This step can be repeated any number of times, the cells being exposed to different factors or the same factors in different concentrations.

A number of substances (exogenous factors) can be used for the Primmg. In particular, combinations, in particular of those mentioned below, cytokme and growth factors, cytokme and transcription factors, cytokme and neurotransmitters, cytokme and hormones, cytokme and gangliosides, cytokme and conditioned media, growth factors and transcription factors, growth factors and neurotransmitters, growth factors and ganglioside growth factors , Growth factors and conditioned media, transcription factors and neurotransmitters, transcription factors and hormones, transcription factors and gangliosides, transcription factors and conditioned media, neurotransmitters and hormones, neurotransmitters and gangliosides, neurotransmitters and conditioned media.

In particular, combinations of cytokms and growth factors can also be used together with transcription factors or neurotransmitters or hormones or gangliosides or condiomeric media. Combinations of cytokms and transcription factors can also be used together with neurotransmitters or hormones or gangliosides or conditioned media. Combinations of cytokms and neurotransmitters can also be used together with hormones or gangliosides or conditioned media. Combinations of growth factors and transcription factors can also be used together with neurotransmitters or hormones or gangliosides or conditioned media. Combinations of growth factors and neurotransmitters can also be used together with hormones or gangliosides or conditioned media. Combinations of growth factors and hormones can also be used together with gangliosides or conditioned media. Combinations of growth factors and gangliosides can also be used together with conditioned media. One or more of the epidermal growth factor (EGF), in particular EGF1, EGF2, EGF3 with the subgroups α and ß, transformmg growth factor (TGF) and ß, LIN-3-Protem, fibroblast growth factor (FGF) are used as growth factors , FGF1 and FGF2, nerve growth factor (NGF), bram-derived neurotrophic factor (BDNF), neurotrophs (NT), in particular NT-3, NT-4, NT-5, NT-6, msulm-like growth factors (IGF ), in particular IGF-1 and IGF-2, glial cell lme-derived neurotrophic factor (GDNF), Neurturm (NTN), Persephm (PSP), vascular endothelial growth factor (VEGF), including their subgroups or factors with a similar effect.

One or more of the group interleukme (IL 1-16), leukemia mhibitory factor (LIF), ciliary neurotrophic factor (CNTF), tumor necrosis factor (TNF), in particular TNF-α, interferons (IFN) are cytokme. especially IFN-α, macrophage mhibitory or stimulation factors, in particular macrophage migration mhibitory factor (MIF), mitochondrial import stimulation factor (MSF) and retinoic acid.

One or more from the group Dopamm, Acetylcholm, GABA, Glutamate, Glycm, Taurm, Prolm, Noradrenalm, Serotonm and Neuropeptides, in particular Substance P and Enkephalm, are used as neurotransmitters.

The neurotransmitters will be used all or in the presence of growth factors and / or cytokms.

Individually or in combination with the substances or combinations of substances mentioned above, hormones such as growth hormones, thyroid hormones (in particular for differentiating the progenitor cells to dopammergenic neurons), steroid hormones or gangliosides, in each case including their derivatives, can be used. In particular, GDNF, LIF and one or more of IL1-11 can be used individually or in combination to generate dopaminergic neurons, in particular the combination IL-1, GDNF, LIF, IL-11, in each case including their subgroups.

The exogenous factors can be used individually or in combination, in each case m concentrations of 25,000 to 0.005 ng / ml, preferably 1000 to 0.1 ng / ml, particularly preferably 100 to 1 ng / ml expansion solution, without being restricted to this

In particular, IL-1 m concentrations of 0.005 to 10 ng / ml, preferably 0.01 to 2 ng / ml, particularly preferably 0.05 to 0.25 ng / ml can be used for differentiation. IL-11 and LIF can be used in concentrations of 0.01 to 100 ng / ml, preferably 0.1 to 20 ng / ml, particularly preferably 0.5 to 2.5 ng / ml. GDNF can be used in concentrations of 1 to 25,000 ng / ml, preferably 1-10 to 5000 ng / ml, particularly preferably 1-100 to 2,500 ng / ml.

The factors can also be used in combination in these concentrations. However, the concentrations to be used are not limited to the values mentioned above and can vary, inter alia, depending on the other factors used.

Partial differentiation through transfection:

Genetic manipulations can also be used, in particular within the framework of the above described but also independently of this. By transfecting the progenitor cells with genes that are relevant in the development of the specific neurons, differentiation into the desired cell type is promoted. Also a temporary expression of these genes, which does not change the genetic material of the cells and No foreign genes after the transplant m infiltrates the host brain determines the further fate.

The development of dopammergenic neurons can in particular be controlled by transfection of genes which encode members of the steroid and thyroid hormone receptor family, such as tyrosine hydroxylase, Nurr-1 and / or Nurr-77 receptors. In particular, genes of the vesicular monoamm transporter or the dopamm transporter can also be used. In general, genes can be used that are specific for dopammerge neurons.

For the selection of cholmergic neurons specific genes can be transferred for these neurons, in particular genes of the nicotmergenic acetylcholre receptor, in particular prasynaptic α and β subunits, in particular α-7, genes of the nerve growth factor (NGF) receptor or cholmesterase.

Genes coding for γ-ammobutyric acid (GA-BA) transporters, genes coding for dopamm receptor, genes coding for glutamate receptor, enkephalm or substance P are of particular importance for striatal neurons.

The corresponding cDNA's with the above-mentioned genes are known from the literature and are available. The transfection is carried out using the standard methods given in the literature. This enables a transient or stable transfection of the progenitor cells.

Selection by subcloning:

Subcloning is particularly suitable for selection. By subcloning, cell cultures can be generated from heterogeneous cell cultures (in this case the neuronal progenitor cells obtained), which consist of a single cell these progenitor cells have arisen (monoclonal or possibly polyclonal cell lines). The heterogeneity of these cells can thus be minimized without the addition of exogenous factors.

The progenitor cells can be subcloned by final dilution of the cell suspension, by micromanipulation or fluorescence-activated cell sorting (“fluorescence-activated cell sorting” (FACS)) after marking the vital cells or by magnetic concentration after marking the cells with supraparamagnetic particles.

The subcloning of the progenitor cells by final dilution of the cell suspension is carried out, for example, by diluting the cell suspension to such an extent that only one cell remains in each culture vessel. The cells thus plated are expanded, whereby monoclonal cell lines are obtained. Mitogenic substances are preferably added to the media (see growth factors given above) in order to achieve multiplication from a single cell. The expansion, differentiation and characterization then takes place as described above for polyclonal progenitor cell suspensions.

The progenitor cells can be subcloned by micromanipulation after fluorescent labeling of the vital cells by staining the living cells with a marker specific to the respective cell population. The labeled cells are then read under a fluorescence microscope using micro-tools (e.g. glass capillaries) so that only labeled cells of one type remain in the culture vessel. The cells plated out in this way are expanded, thereby obtaining cell mines which contain only one cell type.

For expansion, the media are preferably mitogenic Substances added (see growth factors given above) to achieve multiplication from a single cell. The expansion, differentiation and characterization then takes place as described above for the polyclonal progenitor cell suspension. Subcloning using final dilution (see above) can be connected if necessary. As a marker for dopammerge cells, the cells are temporarily transfected, for example, with the gene for the green fluorescent "en anced green-fluorescence protem" (EGFP), which are expressed under the control of specifically dopammerger promoters (Tyros hydroxylase and dopamm transporter promoter). The green glowing cells express the two specific dopammergenic proteins mentioned above and will then be cloned as described for cholmerge cells using the same technique with the Cholm acetyl transferase (ChAT) promoter and for GABAergic neurons with the glutamyl decarboxylase promoter (GAD) or other suitable promoters, furthermore the living cells can be labeled by means of fluorescent antibodies, for dopammerge cells antibodies against the dopamm transporter, for cholmerge

Cells against, in particular, pre-synaptic α and β subunits of the nicotmergenic acetylchol receptor, in particular α7, and for GABAergic cells against the GABA transporter can be used.

The subcloning of the progenitor cells by fluorescence-activated cell sorting can be carried out by means of flow cytometry ("fluorescence-activated cell sortmg"; FACS) after fluorescent labeling of the vital cells by staining the living cells with a marker specific for the respective cell population. The labeled cells are then sorted by means of the FACS device according to the standard procedure specified by the Literaut (for an overview: Orfao and Ruiz-Arguelles, Clm Biochem 1996; 29: 5-9), so that only marked Cells of one type remain in the culture vessel. The cells plated out in this way are expanded, thereby obtaining cell mines which contain only one cell type. For expansion, the media are preferably mixed with mitogenic substances (see growth factors given above) in order to achieve multiplication from a single cell. The expansion, differentiation and characterization then takes place as described above for the polyclonal progenitor cell suspension. Subcloning using final dilution (see above) can be connected if necessary. As a marker for dopammerge cells, the cells are temporarily transfected, for example, with the EGFP gene, which is expressed under the control of specifically dopammerger promoters (tyrosmhydroxylase and dopam transporter promoters). The green glowing cells express the two specific dopammergenic proteins mentioned and will then be cloned as described. For cholmerge cells, the same technique can be used, for example, with the Cholm acetyl transferase (ChAT) promoter, for GABAergic neurons with the glutamyl decarboxylase (GAD) promoter. The living cells can also be labeled using fluorescent antibodies. Antibodies against the dopamm transporter can be used for dopammerge cells, for cholmerge cells against prasynaptic α and β subunits of the nicotmergenic acetylcholre receptor, in particular α7, and for GABAergic cells against the GABA transporter.

The progenitor cells are subcloned by magnetic concentration after marking the cells with supraparamagnetic particles by magnetically marking the living cells with a marker specific to the respective cell population. Various particles are available (Basic microbeads - dextran coated with free amines, 50nm, Miltenyi Biotech; Ammo / Carboxy beads, 110-140 nm, Immunicon Corp .; Streptavidm / Biotm coated, Miltenyi Biotech or Immunicon Corp.). These particles can be loaded with ligands for specific surface protests (eg dopamine D2 receptors, dopamm transporters or other transporters, nicotinic acetylcholme receptors). Cells that bind to these particles can be connected using magnetic columns.

The plated cells are expanded, thereby obtaining cell lines containing only one cell type. For expansion, the media are preferably mixed with mitogenic substances (see growth factors given above) in order to achieve multiplication from a single cell. The expansion, differentiation and characterization then takes place as described above for the polyclonal progenitor cell suspension. Subcloning using final dilution (see above) can be connected if necessary. The labeling of the living cells is carried out using antibodies which have been coupled with magnetic “microbeats”. Antibodies against the dopamm transporter can be used for dopammerge cells, for cholmerge cells against prasynaptic α and β subunits of the nicotmergenic acetylchol receptor, in particular α7, and for GABAergic cells against the GABA transporter. The use of low molecular weight ligands of cell type-specific proteins is also possible. Ligands for the dopamm D2 receptor (benzamide or spiperondentate) or the dopamm transporter (cocaine derivatives) would be suitable for dopammerge neurons.

In all of these methods, the cells are subcloned at a stage in which the greatest possible differentiation has taken place without reducing the ability of the cells to divide, that is to say after a primming, genetic manipulation, change in the atmosphere or treatment with exogenous factors.

The steps of partial differentiation, selection (cloning) and expansion described above can be combined and repeated if necessary.

General procedure

After the preparation of the brain tissue, the homogenization takes place. Advantageously, a proteolytic enzyme, in particular a Ser protease such as Trypsm, preferably in a concentration of 50 to 500 mg / ml, is added to the tissue in order to loosen the tissue bond.

The tissue thus prepared is then advantageously mixed with a DNase solution, which preferably contains a concentration of 0.5 to 20 mg, preferably 2 to 6 mg, of DNase per 100 ml of solution. DNase I is preferably used. The DNase solution is incubated for 2 to 30 minutes, preferably about 10 minutes, to digest extracellular DNA, as this can impair cell survival and homogenization.

Subsequently, the digested tissue parts are homogenized by repeated opening in a glass pasteup pipette.

Then the tissue is mixed with an effective amount of an expansion medium to multiply the progenitor cells and expanded in culture bottles, as is known from the literature given.

In addition to the expansion-demanding (mitogenic) factors, the expansion medium can contain 10 to 60%, preferably 30 to 45% F-12 medium, 30 to 75%, preferably 45 to 70% Dulbecco's Modified Eagle's Medium (DMEM; glucose-free or with low levels or 17 high glucose content), effective amounts of a suitable antibiotic, such as penicillin or streptomycm, m in a concentration of 50 to 250 IU / ml for penicillin and 50 to 250 μg / ml for streptomycm, preferably 150 IU / ml for penicillin and 150 μg / ml for Streptomycm included.

The expansion media can furthermore one or more substances from the group Transferrm, biogenic nurse, especially diamines such as e.g. 1, 4-Dιammbutan (Putrescm), bactericidal reducing agents such as sodium selenite, progestogens, in particular progestogens such as progesterone or progesterone-like active ingredients, and insulin. Alternatively, various supplements extracted from serum, such as the commercially available B-27 supplement (Gibco) can be used.

The expansion can be carried out in a modified atmosphere with a reduced atmospheric oxygen content. For this purpose, the cells of a corresponding chamber are stored, in which an exact oxygen concentration is guaranteed. Depending on the desired cell type, the oxygen content is varied between 1% and 10%.

Mitogenic factors in particular can be added to the medium for the cell cultures to be cloned here. The factors mentioned above are advantageously used for this. The subcloning is also preferably carried out under a changed atmosphere. It has proven to be advantageous to adjust the oxygen concentration very individually to the isolated cells, in particular to values between 1% and 15%.

After sufficient expansion of the cell clones, the selection and partial differentiation of the cells into transplantation-free cell cultures takes place. The transfection of the cells with genes can be carried out with the addition of suitable amounts, for example 0.5 μg of plasmid DNA per ml content of a culture vessel and 3 μl per μg DNA from TransFast (Pro mega) solution to 1 ml of the expansion medium in accordance with the provisions of Suppliers. This solution can be incubated at 37 ° C and then added to the tissue sample. To identify the differentiated neurons, DNA and lipofectm can be incubated and then differentiated in medium I. The cells can then be harvested and processed in the usual way.

The measurement of the proliferation by incorporating [ ³ H] thymidm and the protein determination as well as the measurement of the proliferation and vitality by flow cytometry are carried out according to customary methods.

To differentiate the progenitor cells, the cells can be applied in vitro by plating on poly-L-Lysm-coated cover strips or 48-hole plates in neurobasal medium (Gibco). The media can be mixed with FCS, cytokmas and / or stπatal-conditioned media. For example, the cytokme IL-1B, IL-11, LIF, GDNF or other exogenous factors as described under the section "Primmg" can be used. The cells are kept for 7 to 10 days at 37 ° C. in a humidified atmosphere before Fixation and further examinations differentiated.

The functional integrity of the neurons, e.g. DA and GABA neurons, can be done by measuring the uptake of kicked neurotransmitters. After pram incubation for 10 minutes an incubation buffer containing 100 μM Pargylm,

ImM ascorbate and 2mM ß-alanm (and to determine the non-specific uptake: 3μM GBR12909 and ImM 2, 4-diammo-n-butyric acid; DABA) can contain 50 nM [ ³ H] DA, [ ³ HjCholin or [ ³ H] GABA be added for 15 minutes at 37 ° C. Uptake can be stopped by washing the plates with cold PBS and the remaining radioactivity of the cell lysate can be measured by using liquid scintillation counting. The specific intake can be determined as the difference between the intake in the absence (total) and the intake in the presence of GBR12909 and DABA (non-specific).

The invention is described below by way of example.

Exemplary embodiment

To obtain progenitor cells for the production of human neuronal cells of a certain type or a corresponding transplantable neuronal cell material, the brain of a human, five to eleven week old fetus is used, the meninges of which have been removed. The brain substance is prepared with cooling, for example in a Petri glass dish on ice. The parts of the brain from which the progenitor cells are to be obtained are prepared with two scalpels, the periventricular sections also being prepared. The prepared brain parts are placed in sterile plastic tubes with 12 to 14 ml of a dissection solution on ice.

The dissection solution contains 98% calcium and magnesium free Hank s buffered salt solution (HBSS), 150 IU. / ml Penicil- lιn / 150 μg / ml Streptomycm and 4500 mg / 1 D-glucose.

To expand the neuronal progenitor cells, the dissection solution containing the neuronal cell material is centrifuged and the supernatant is aspirated. The separated tissue with 1 ml trypsin solution (250 mg Trypsm m 100 ml HBSS, sterile filtered and stored in 1 ml samples at - 20 ° C freeze) added and shaken. The solution is incubated for 30 minutes at room temperature. The sample is then centrifuged and the supernatant aspirated from the tissue.

The pellet DNAse solution thus obtained (4 mg DNase I, 15 mg soybean trypsin inhibitor, 188.2 mg MgS0 ₄ to 100 ml HBSS, sterile filtered and frozen for storage in 2 ml samples at -20 ° C.) is then added and shaken .

The solution is incubated for ten minutes at 37 ° C., then centrifuged and the supernatant is suctioned off from the tissue.

The tissue is then mixed with 3 to 5 ml of expansion medium I or expansion medium II for cell growth.

The expansion medium I consists of 32% F-12 medium, 65% Dulbecco ^'s Modified Eagle ^{^} s medium (DMEM) (4500 mg / 1 D-glucose), 2% B-27 supplement solution (company Gibco), 150 IU . / ml penicillin / 150 μg / ml streptomycin and the growth factors EGF (20 ng / ml), optionally also EGF (20 ng / ml) and FGF 2 (20 ng / ml).

The expansion medium II contains 45.1% F-12 medium, 45.1% DMEM, 1 ml transferrin stock solution (100 mg transferrin to 10 ml HBSS), 1 ml putrescine stock solution (consisting of a 126 mg putrescin hydrochloride to 100 ml HBSS) accordingly a putrescin concentration of 60 μM), 1 ml penicillin / streptomycin, 200 μl Na selenite stock solution (1 mg sodium selenite, 11.6 ml H ₂ 0 0.3 ml of this solution to 9.7 ml HBSS gives the Stock solution), 200 μl progesterone stock solution (1 mg progesterone to 3.2 ml ethanol, 50 μl of the ethanol solution to 4.95 ml HBSS gives the stock solution), 200 μl insulin stock solution (100 mg Insulin on 10 ml HC1, 0.01 N) and the growth factors EGF (20 ng / ml) and FGF 2 (20 ng / ml).

The expanded tissue is homogenized, for example with an Eppendorff pipette, and the cell number is determined with a hemocytometer. The cell suspension is diluted with expansion medium I or II to a cell number of approximately 300,000 cells / ml. 8 ml of this cell suspension (corresponding to about 2.5 million. Cells) are placed on a 25 cbm bottle and the cells were cultured with an atmosphere containing 5% C0 _2/95 air at 37 ° C for about a week.

The cells are reacted once or twice a week in fresh expansion medium I or II, for which the cells are detached in the bottle and transferred to plastic tubes and then centrifuged, the supernatant is aspirated and 2 ml of the fresh expansion solution I or II are added and then homogenized . The homogenized solution is divided into several samples, e.g. 3 to 6 samples, divided, transferred to new bottles and mixed with 8 ml of the expansion solution mentioned above.

The expanded progenitor cells as described above can be deep-frozen for storage at -80 °. For this purpose, the cells are detached from the culture bottle with the trypsin solution described above and the cell suspension is centrifuged with 100 g for 10 minutes. The pellet obtained after pouring off the supernatant is taken up in the expansion solution I or II and the cell suspension is diluted with the corresponding expansion solution to a cell number of 2 to 4 × 10 ⁶ per ml. The cell suspension obtained in this way is temporarily stored on ice and then 0.5 ml of the cell suspension in a freezer tube pre-cooled at -80 ° C. with the addition of 0.5 ml of a freezing medium (consisting of 50% fetal calf serum, 20% dimethyl sulfoxide and 30% of the expansion solution I. or II) filled. The sealed tubes are frozen in a styrofoam box at -80 ° C.

As a control, the samples are thawed the next day or later and, if there is sufficient cell activity, the remaining samples frozen at -80 ° C are sealed in a freeze-proof film and sealed in liquid nitrogen.

To control the frozen cell samples, the thawed cells are multiplied, differentiated and characterized using the methods specified above, which depend on the cell type. The frozen progenitor cells, which have sufficient cell characteristics, are stored and used as a transplant.

The progenitor cells prepared in this way can be stored in liquid nitrogen for an unlimited period.

For the subsequent thawing of the cell samples, the freezing tubes are taken directly from the liquid nitrogen and completely thawed in a water bath at 37 ° C. The tube is then disinfected with 70% ethanol. Transfer the cell suspension into 15 ml plastic tubes and add 9 ml of expansion medium I or II slowly and under rubble. The resulting suspension is centrifuged at 100 g for ten minutes, the supernatant is poured off and the cell pellet is taken up in 6 ml of expansion medium I or II. The sample is then transferred to a 50 ml culture bottle and incubated in an atmosphere with 5% CO ₂ /95% air at 37 ° C for about a week. The cells are then expanded further as described above.

The progenitor cells can be differentiated using freshly expanded or thawed samples. For this purpose, the cells are rinsed from the culture bottle, centrifuged and taken up in a suitable recording medium, so that a cell count of approximately 20,000 to 100,000 cells / ml results.

The compositions of three suitable recording media are given below by way of example.

The recording medium I consists of 45.1% F-12 medium, 45.1% DMEM, 1 ml Transferrm stock solution, 1 ml Putrescm stock solution (results in 60 μM), 150 IU / ml penιcιllm / 150 μg / ml Streptomycm, 200 μl Na Selemt stock solution, 200 μ 1 progesterone stock solution and 200 μl Insulm stock solution.

The compositions of the stock solutions mentioned correspond to the stock solutions of the above-described preparation of the expansion medium II.

The recording medium II contains 44.5% F-12 medium, 44.5% DMEM, 150 I.U./ml Penιcιllm / 150 μg / ml Streptomycm and 10% fetal calf serum (not heat activated).

The recording medium III contains 98% of a neurobasal medium (Gibco company), 1% N-2 supplement (Gibco company) and 150 I.U./ml penιcιllm / 150 μg / ml Streptomycm.

The differentiation media containing the cell material are IL-1 (100 pg / ml), IL-11 (1 ng / ml), GDNF (1 μg / ml) and LIF (1 ng / ml) together or these substances in the The stated concentrations are added individually.

The samples are incubated for 7 to 21 days in an atmosphere with an oxygen content of 2%.

The cells obtained are then subcloned selected in an atmosphere with 5% oxygen, after which the steps of expansion and differentiation described above are repeated.

The resulting cell suspensions are taken up in a phosphate-buffered salt solution for transplantation.

In each case, preparations were obtained from dopammergenic neurons that are practically free of glial cells. The cells can then be transplanted.

Suspensions of determined progenitor cells can also be produced, which differentiate partially or completely from the human brain without further treatment after implantation or upon contact with another cell medium or carrier material. If necessary, a final differentiation of the neurons can also follow the method steps mentioned.

Claims

Neural cell material, use as a graft, and method of making the same

1. Isolated neuronal cell material, due to the fact that the cell material does not contain any physiologically effective portions of immune-competent glial cells.

2. Cell material according to claim 1, d a d u r c h g e k e n n z e i c h n e t that the cell material contains practically exclusively dopammergic neurons and / or cholmergic neurons and / or GABAergic striatal and / or serotonergic neurons or cells which can differentiate into these neurons.

3. Cell material according to claim 1, so that the cell material consists essentially of dopammergenic neurons or essentially contains precursors of these cells which can differentiate these neurons.

4. Cell material according to claim 1, so that the cell material consists essentially of cholmergenic neurons or essentially contains cells which can differentiate these neurons.

5. Cell material according to claim 1, characterized in that the cell material consists essentially of GABAergic striatal neurons or essentially contains cells which can differentiate these neurons m.

6. Cell material according to claim 1, which also means that the cell material consists essentially of serotonergic neurons or essentially contains cells which can differentiate into these neurons.

7. Cell material according to any one of claims 1-6, d a d u r c h g e k e n n z e i c h n e t that the cells are mammal, especially human, cells.

8. Cell material according to claim 7, d a d u r c h g e k e n n z e i c h n e t that the cell material was obtained from progenitor cells.

9. Transplantable neuronal cell material that was obtained from cell material according to one of claims 1-8.

10. Progenitorzellmie from monoclonal mammalian, especially human progenitor cells, which differentiate exclusively or predominantly into neuronal cells by adding suitable differentiation agents.

11. A process for producing a growth-capable cell culture from progenitor cells, comprising the following process steps:

- removal of brain parts from a mammal,

- selection of progenitor cells,

- multiplication of progenitor cells

- partial differentiation of progenitor cells

- Repeat one or more of the steps of expansion, selection and / or partial differentiation if necessary.

12. The method according to claim 11, characterized in that the method is a selection of Progenitor cells covered by subcloning.

13. The method according to claim 12, so that the subcloning is carried out by one or more methods carried out in a suitable time sequence from the following group:

- subcloning by final dilution,

- Subcloning by micromanipulation of marked vital cells

- subcloning by fluorescent cell sorting of marked vital cells,

- Subcloning by magnetic concentration of magnetically marked cells.

14. The method according to any one of claims 11 - 13, characterized in that at least one process step from the group subcloning, Primmg and / or treatment with exogenous factors is used, which is carried out in each case under an atmosphere with reduced or increased oxygen and / or nitrogen content or simulates the changes that can be induced by a changed oxygen and / or nitrogen content.

15. The method according to claim 14, so that the oxygen content of the atmosphere is less than 10% by volume, preferably less than 5% by volume.

16. The method according to any one of claims 14-15, that the changes in the reduced oxygen and / or nitrogen content in the atmosphere are simulated by other treatments.

17. The method according to any one of claims 11 - 16, characterized characterized in that a partial differentiation takes place by at least one process step from the group Primmg and genetic modification.

18. The method according to any one of claims 11 - 17, characterized in that a partial differentiation by Primmg by means of treatment with suitable cytokms and / or growth factors and / or transcription factors and / or neurotransmitters and / or hormones and / or gangliosides in effective amounts and subsequent Incubation is carried out under suitable conditions for a sufficiently long period of time.

19. The method of claim 18, d a d u r c h g e k e n n z e i c h n e t that the partial differentiation by Primmg with the addition of one or more of the

Growth factors from the group EGF, FGF, GNDF, TGF α and ß, LIN-3 protein, NGF, BDNF, NT, CNTF, PDNF, IGF, VEGF, including their subgroups.

20. The method according to claim 18 or 19, characterized in that for multiplication and / or differentiation, one or more cytokines from the group LIF, interleukme (IL 1-16), TNF, interferons, macrophage inhibitor-stimulating factors, in particular MIF, MSF, retinoic acid, be added.

21. The method according to any one of claims 18 - 20, characterized in that used for partial differentiation by Primmg em or more of the neurotransmitters from the group Dopamm, acetylcholm, GABA, glutamate, glycine, Taurm, Prol, Noradrenalm, Serotonm, substance P and Enkephalm become.

22. The method according to any one of claims 11-21, characterized characterized in that monoclonal progenitor cell lines are used for the partial differentiation of the progenitor cells.

23. The method according to any one of claims 11-22, since it is known that the expansion of the progenitor cells takes place under hypoxic conditions and / or with the addition of exogenous factors.

24. Cell culture, so that the cells were produced by a method according to claims 11-23.

25. Transplantable neuronal cell material, so that the cell material is obtained from a viable cell culture according to claim 24.